Genetically engineered glutaminase and its use in antiviral and anticancer therapy

ABSTRACT

DNA encoding a therapeutically suitable glutaminase has been molecularly cloned. This allows one to obtain a polypeptide which is a therapeutically suitable glutaminase free of contaminating endotoxin. It has been found that this polypeptide is a potent anti-viral agent and when coupled to an anti-tumor monoclonal antibody is a potent anti-cancer agent. The glutaminase of the present invention is particularly useful for treating lung, breast and colon cancer cells and in the treatment of HIV-infected cells.

This application is a national stage application of PCT/US92/10,421 fileDec. 4, 1992.

This invention was made with government support under Contract No.97-F156500-000 awarded by the Defense Advanced Research Projects Agency(DARPA). The government has certain rights in this invetion.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a DNA coding for a therapeuticallysuitable glutaminase, a polypeptide which has the activity of atherapeutically suitable glutaminase, as well as their use in antiviraland anticancer therapy.

BACKGROUND OF THE INVENTION

Use of glutaminase to deplete glutamine in tumor-bearing hosts offers anattractive method for attacking cancer cells. Glutamine occupies animportant role in the biosynthesis of a large number of cellularmetabolites. Compared with normal tissues, some neoplasms have beenshown to operate at a marginal level of glutamine availability becauseof decreased synthesis and stepped-up utilization (Levintow, 1954, J.Natl. Cancer Inst. 15:347-352; Roberts, et al., 1960, Amino Acids,Proteins and Cancer Biochemistry (J. T. Edsall, ed.), Academic Press.New York, N.Y. pp. 121-145; Weber, G., 1983., Cancer Res. 43:3466-3492;Sebolt, et al., 1984, Life Sci. 34:301-306). Experiments have revealed anegative correlation between glutamine content and the growth rate oftransplanted rat hepatoma tumors. The in vivo concentration of glutaminein hepatoma 3924A was found to be 9-fold lower (0.5 mM) than in liver(4.5 mM) and lower than in any other rat tissues (2 to 5 mM) (Weber,1983, Cancer Res. 43:3466-3492). In recent years accumulated dataindicate that glutamine may be an important fuel source of cellularenergy in a variety of neoplasms, including hematopoietic tumors,hepatomas, Ehrlich carcinoma, and HeLa cells (Abou-Khalil, et al., 1983,Cancer Res. 43:1990-1993; Kovacevic, et al., 1972, J. Biol. Chem.33:326-333; Kovacevic, 1971, Biochem. J. 125:757-763; Reitzer, et al.,1979, J. Biol. Chem. 254:2669-2676).

L-asparaginase, the first enzyme to be intensively studied as anantitumor agent in man, is highly effective in the treatment of acutelymphoblastic leukemia. This enzyme, however, has little or no activityagainst any other neoplasms in humans. The enzyme glutaminase hasactivity against a much broader range of cancers than asparaginase.

Several mammalian and microbial glutaminase and glutaminase-asparaginaseenzymes have been purified and characterized. Of these Pseudomonas 7Aglutaminase-asparaginase appears to be best suited for therapeutic usebecause of its low K_(M) for glutamine (micromolar range), goodstability and activity in a physiological milieu, and long plasmahalf-life in tumor-bearing hosts (Roberts, 1976, J. Biol. Chem.251:2119-2123, and Roberts, et al., 1979, Cancer Treat. Rep.63:1045-1054).

The known mammalian glutaminase enzymes are not suitable for use astherapeutic agents because of their high K_(M) values (millimolarrange), and their requirement for phosphate esters or malate foractivation. The E. coli glutaminases (A and B) are also unsuited fortherapeutic use because of their high K_(M) values (millimolar range),low activity at physiological pH (glutaminase A), or requirement forspecial activating substances (glutaminase B).

Pseudomonas 7A glutaminase-asparaginase is composed of four identicalsubunits with a molecular weight of approximately 35,000. Active enzymesedimentation studies indicate that the catalytic activity is associatedwith the tetramer; no smaller active species are observed (Holcenberg,et al., 1976, J. Biol. Chem., 251:5375-5380). The purified enzyme has aratio of glutaminase to asparaginase activity of approximately 2:1.Binding studies with C¹⁴-labelled analogs of glutamine(6-diazo-5-oxo-L-norleucine; DON) and asparagine(6-diazo-5-oxo-L-norvaline; DONV) suggest that the two analogs may reactpreferentially with hydroxyl groups at two different sites on theprotein, and that the two binding sites act cooperatively as part of theactive site (Holcenberg, et al., 1978., Biochemistry 17:411-417).

Pseudomonas 7A glutaminase-asparaginase was shown to have considerableantineoplastic activity against a variety of rodent leukemia (L1210,C1498, EARAD/1), ascites tumors (Taper liver, Ehrlich carcinoma, meth Asarcoma, S 180) and certain solid tumors (Walker 256 carcinosarcoma, B16melanoma). Additionally, antagonism of glutamine by glutamine analogsand glutaminase was found to be strongly inhibitory to human colon,breast and lung carcinomas growing in athymic mice (McGregor, 1989,Proc. Amer. Assoc. Cancer Res. 30:578; Roberts, 1979, Cancer Treat. Rep.63:1045-1054; Ovejera, 1979, Cancer Res. 39:3220-3224; Houchens, 1979,Cancer Treat. Rep. 63:473-476; Duvall, 1960, Cancer Chemother. Rep.7:86-98).

An important characteristic of glutaminase therapy is that resistantstrains do not develop after repeated treatments with this enzyme(Roberts, 1979, Cancer Treat. Rep. 63:1045-1054). Treatment withglutaminase was also shown to delay development of resistance againstmethotrexate (Roberts, 1979, Cancer Treat. Rep. 63:1045-1054).

A bioactive glutaminase-asparaginase has been shown to inhibit mouseretroviral disease. Glutamine depletion strongly inhibits thereplication of Rauscher murine leukaemia retrovirus (RLV) in vitro.Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depletingglutamine and asparagine for prolonged periods, was used to determinethe therapeutic effectiveness, of glutamine depletion in mice infectedwith RLV or Friend virus. During PGA treatment of viremic animals, serumreverse transcriptase activity fell to control levels and infectedanimals did not develop splenomegaly. The therapeutic results obtainedwith PGA compare favorably with those of azidothymidine givenintraperitoneally at 30 mg/kg/day (Roberts, 1991, Journal of GeneralVirology, 72:299-305).

Despite the promise of glutaminase as a therapeutic agent, there arecurrently no therapeutically useful glutaminases available which can beproduced cheaply and with little or no contamination by othersubstances, for example by endotoxins of a host microorganism. Moreover,a suitable enzyme is not available in quantities which are large enoughto allow for wide-spread clinical trails.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method of inhibiting thereplication of HIV in infected cells.

It is another object of the invention to provide a method of inhibitingthe growth of cancer cells.

It is yet another object of the invention to provide an E. coli cellwhich comprises a therapeutically suitable glutaminase.

It is still another object of the invention to provide a DNA moleculeencoding a therapeutically suitable glutaminase.

It is an object of the invention to provide a therapeutically suitableglutaminase free of Pseudomonas endotoxin.

It is another object of the invention to provide methods of treatingtransformed cells in a body.

It is still another object of the invention to provide a therapeuticcomposition for treating neoplastic cells.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment a method ofinhibiting the replication of HIV in HIV-infected cells is provided. Themethod comprises administering a therapeutically suitable glutaminase toHIV-infected cells in an amount sufficient to inhibit replication of HIVin said cells.

In another embodiment of the invention a method of inhibiting the growthof cancer cells is provided. The method comprises administering a boundcomplex to tumor cells which express a tumor-associated antigen, theamount of said complex administered being sufficient to inhibit DNAsynthesis in said tumor cells. The complex comprises: (a) atherapeutically suitable glutaminase and (b) an antibody immunoreactivewith a tumor associated antigen.

In yet another embodiment of the invention an E. coli cell is providedwhich comprises a Pseudomonas 7A glutaminase-asparaginase gene.

In still another embodiment of the invention an isolated and purifiedDNA molecule is provided. The molecule comprises a nucleotide sequencecoding for a therapeutically suitable glutaminase.

In one embodiment of the invention a cell-free preparation of atherapeutically suitable glutaminase is provided. The preparation isfree of Pseudomonas endotoxin.

In another embodiment of the invention a method of treating transformedcells in a body is provided. The method comprises: administering aplasmid comprising the nucleotide sequence of SEQ ID NO:1, said sequenceunder the transcriptional control of a tissue-specific promoter, saidplasmid coated with poly-L-lysine covalently linked to a tissue-specificligand.

In still another embodiment of the invention a therapeutic compositionis provided. The composition comprises: a complex comprising atherapeutically suitable glutaminase and an antibody which is specificfor a tumor-associated antibody.

In another embodiment of the invention a method of treating atumor-bearing patient is provided. The method comprises the steps of:obtaining tumor infiltrating lymphocytes from a tumor-bearing patient;transfecting said tumor infiltrating lymphocytes with a vector whichcauses expression of Pseudomonas 7A glutaminase in human cells; andadministering said transfected tumor infiltrating lymphocytes to thepatient to supply said tumor with Pseudomonas 7A glutaminase.

In still another embodiment of the invention, a method of treating atumor-bearing patient is provided which comprises the following steps:obtaining tumor infiltrating lymphocytes from a tumor-bearing patient;complexing said tumor infiltrating lymphocytes with a vector comprisingthe nucleotide sequence of SEQ ID NO:1, said vector causing expressionof Pseudomonas 7A glutaminase in human cells; and administering saidcomplex of lymphocytes and vector to the tumor-bearing patient to supplysaid tumor with Pseudomonas 7A glutaminase.

The present invention thus provides the art with new and usefulanti-tumor and anti-viral therapeutic agents, as well as tools formaking them and methods for using them.

The cloning and expression of the gene that encodes Pseudomonas 7Aglutaminase-asparaginase in E. coli increases the glutaminase producedper liter of culture at least 12-fold, relative to the yield inPseudomonas 7A. This markedly reduces the production cost of glutaminaseand enables widespread clinical trials. Additionally, by producing theglutaminase in E. coli contamination of the antitumor drug by highlytoxic Pseudomonas endotoxin is avoided.

BRIEF DESCRIPTION OF THE INVENTION

FIGS. 1A-1D show the nucleotide and deduced amino acid sequence of thePseudomonas 7A glutaminase gene (SEQ ID NOS. 1 and 2. The top strand ofthe coding DNA sequence is shown from 5′-3′. The numbers shown indicatenucleotide base pairs. The deduced peptide sequence is shown below theDNA sequence. The engineered N-terminal methionine residue is not shown.

FIGS. 2A-2B depict the sequencing strategy for the Pseudomonas 7Aglutaminase gene. FIG. 2A: Map of the P7A glutaminase showing selectedrestriction sites, the shaded area depicts the region encoding theactual gene product. Hatch marks represent 100 bp. Arrows below thisfigure show the approximate positions and orientations of sequencingprimers with their accompanying names. The arrows with stops indicatethe extent and direction of individual sequencing experiments. FIG. 2B:Names, sequences, and coordinates of sequencing primers are shown (SEQID NOS. 3-11). Numbering is from the AAG encoding the N-terminal lysineresidue.

FIG. 3A depicts recombinant constructs with P7A glutaminase. “Ap^(r)” isa β-lactamase gene, conferring ampicillin resistance. “T” represents atranscriptional terminator. “Ptac” is the promoter. “ori” is the pBR322origin of replication.

FIG. 3B shows the construction of a P7A glutaminase over-expressingplasmid.

FIG. 4 shows denaturing polyacrylamide gel electrophoresis of crudeextracts. Lane 1 shows an uninduced control and lanes 2-4 showinductions in whole cell extracts. The arrow indicates the position ofthe induced glutaminase band.

FIG. 5 depicts the induction by IPTG of PGA expression in E. colicontaining pME18.

FIG. 6 shows hybridization of heterologous DNA to Pseudomonas 7Aglutaminase sequences. Lane 1, Pseudomonas 7A; lane 2, Pseudomonasaeruginosa; lane 3, Achromobacter sp.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that glutaminase enzymes canbe molecularly cloned in host organisms, despite the obstacle of hostcell toxicity. Glutaminase activity must be strictly regulated in thisprocess, because it is toxic to the host cells. Applicants have foundthat by means of a promoter which must be induced to express adownstream gene, as well as by using transcriptional terminators both 5′and 3′ to the gene, that the glutaminase activity in the host cell canbe controlled to a sufficient extent for the host cells to survivewithout loss of the DNA which encodes glutaminase. When the molecularclones are expressed in desirable host cells, glutaminase can beproduced without contamination by endotoxins. It is a further finding ofthe present invention that glutaminase has inhibitory activity againsthuman immunodeficiency virus (HIV) replication in infected cells.Moreover, it has been found that when glutaminase is complexed withanti-tumor antibodies and administered to tumor cells, that the growthof the tumor cells is inhibited to an extent far exceeding theinhibition by either glutaminase or antibody alone.

Glutaminase enzymes according to the present invention aretherapeutically suitable if they display high enzyme activity atphysiologic pH, i.e., between about pH 6.5 and 8.5. Therapeuticallysuitable glutaminase enzymes must have a low K_(M), i.e., between 10⁻⁶and 10⁻⁴ M. Additionally desirable properties of glutaminase enzymes fortherapeutic use include:

1. High stability at physiologic pH.

2. Retains high activity and stability in animal and human sera andblood.

3. Cleared slowly from the circulation when injected into animals orhumans. A plasma half-life (t_(½)) for glutaminase greater than sixhours in mice and sixteen hours in humans is desirable.

4. Not strongly inhibited by the products of the reaction it catalyzesor by other constituents normally found in body fluids.

5. Does not require cofactors or prosthetic groups that can easilydissociate from the enzyme.

6. Narrow substrate specificity.

7. Effective irreversibility of the enzymatic reaction under physiologicconditions.

8.Available from an organism that contains low levels of endotoxin.

9. Low immunogenicity.

A number of amino acid-degrading enzymes that do not exhibit antitumoractivity also fail to meet at least one of these criteria. For instance,E. coli glutaminase has a pH optimum of 5 and essentially no activity atphysiologic pH. An ineffective form of E. coli asparaginase has a K_(M)over 1 mM. Asparaginase enzymes from yeast, Bacillus coagulans, andFusanium tricinctum all have excessively rapid clearance rates in mice.

The nucleotide sequence of one such therapeutically suitable glutaminasegene which was cloned is shown in SEQ ID NO: 1. It is derived from theorganism Pseudomonas 7A (P7A). The intact coding region encompasses 1008base pairs and encodes a continuous polypeptide sequence of 336 aminoacids (not including a 24 amino acid putative signal sequence). TheC-terminus is punctuated by tandem stop codons and a putativetranscriptional terminator.

The P7A glutaminase sequence which is disclosed here can be used toidentify similar sequences encoding similar proteins. (See Watson, J. D.et al., in “Molecular Biology of the Gene.” Benjamin/Cummings PublishingCompany Inc., Menlo Park CA, Vol. I, p. 608 (1987)). For example,Southern hybridization experiments can be carried out in whichprokaryotic or eukaryotic organismal DNA is probed with all or part ofthe glutaminase gene of the present invention. Typically probes containat least about 15 bases of the glutaminase sequence in order to ensurethat other non-related sequences do not hybridize. Sufficiently hightemperature and low salt concentrations further reduce hybridization tonon- related sequences. Using such techniques, homologous genes havebeen found to dnaA of E. coli in Pseudomonas putida (Ingmer and Atlung,Mol. Gen. Genet. 232, 431 (1992)) and to ras in a variety of eukaryoticorganisms (Matsui, Gene 76:313 (1989) and Hori, Gene 105:91 (1991)).There is a high probability that DNA sequences that hybridize to the P7Aglutaminase DNA represent genes encoding enzymes of similar function.Genes which are isolated by this technique can be expressed, and theenzymes can be tested to determine if they share the desirablecharacteristics identified for the P7A glutaminase.

Probes contemplated by the present invention may be designed, as isknown in the art, using the precise nucleotide sequences disclosed herefor the P7A glutaminase gene, or based on the amino acid sequence of theenzyme. Thus for some purposes it may be desirable to employ degenerateprobes, i.e., a mixture of probes whose sequences encode the same aminoacids but contain different codons. Use of such probes should allow abroader range of homologous genes to be identified.

Using the DNA sequence of the P7A glutaminase (PGA), it is now possibleto obtain other glutaminase genes from other organisms usingcomplementation cloning. This is a technique which can be used even whenthere is no cross-hybridization or immunological cross-reactivitybetween two glutaminase genes or proteins. See Kranz, et al., Proc.Natl. Acad. Sci. 87:6629 (1990). Generally, the target organism ismutagenized and mutants are selected for an inability to utilizeglutamine as a carbon and/or nitrogen source. Transformation with theP7A glutaminase should restore glutamine utilization in some of theselected mutants. These organisms should contain mutations in a genehomologous to PGA. Reversion of this mutant phenotype by introduction ofDNA isolated from wild-type organisms can then be used as an assay toscreen for the PGA homolog.

Provided the amino acid sequence of the glutaminase gene from P7A, asshown in SEQ ID NO:2, antibodies can routinely be obtained. These can beraised using peptide fragments or the complete protein as immunogens.The antibodies can be polyclonal or monoclonal, as is desired for theparticular purpose. Antibodies can be used for screening strains forrelated enzymes, for quantitating the amount of enzyme present in acell, and for detecting molecular clones from a library of clones.

The glutaminase genes according to the present invention can be readilymodified to increase their compatibility with the host organism. Forinstance, codon usage varies from one organism to another. Therefore, itmay be desirable in order to increase expression efficiency of theglutaminase, to alter the codons to conform to the codon usage patternof the host. Such changes would not alter the amino acid sequence of theglutaminase but only the gene sequence. Such changes can be accomplishedby any means known in the art, for example, oligonucleotide-directedmutagenesis can be used to introduce changes into a gene sequence.Alternatively, the entire gene can be synthesized.

Natural glutaminase contains a secretion signal, i.e., an N-terminalamino acid sequence of about 20 amino acids which is responsible forsecretion through the cell membrane to the periplasmic space. Under someconditions, it may be beneficial to include a signal sequence in aglutaminase expression construct. The natural signal sequence may beused, or other signal sequences may be grafted onto the matureglutaminase sequence, to accomplish secretion of the enzyme. Use of asignal sequence may be advantageous for the expression of glutaminase,because it may diminish the toxic effect on the host cell. One signalsequence which may be used is the E. coli ompT signal. This signal, likeothers, is well known in the art. Secretion of glutaminase from the hostcell may facilitate purification of the enzyme and should lead to theformation and recovery of authentic glutaminase.

Inducible promoters are desirable for expression of glutaminase becauseof the enzyme's inherent toxicity to living cells. Some induciblepromoters which may be used are lac, tac, trp, mal, and P_(L). Choice ofa promoter is within the skill of the art.

Transcriptional terminators are also desirable both 5′ and 3′ to theglutaminase gene to prevent “read-through” expression. Many terminatorsare known and can be used. For a review see Watson, J. D. et al., inMolecular Biology of the Gene, Benjamin/Cummings Publishing Co., MenloPark, CA, Vol I, pp. 377-379 (1987). One terminator which Applicantshave found useful is that of the T7 phage.

Modifications of the glutaminase gene were made for ease of productionof enzyme in E. coli. In one such modified gene methionine is added atthe N-terminus of the mature protein. In another such modified genemethionine, asparagine, and serine were added at the N-terminus of themature protein. Neither of these additions destroyed enzyme activity orsubstrate specificity. Other similar changes are contemplated within thescope of the invention which do not significantly affect enzymefunction.

According to the practice of the present invention it may be desirableto make modifications to the structure of glutaminase in order toimprove its therapeutic properties or the ease of producing it. Forexample, it may be desirable to eliminate portions of the protein, bypremature truncations or targeted deletions, to eliminate portions whichare not essential for enzymatic function. A smaller protein may providean improved therapeutic index by virtue of increased permeability intotumor masses, for example. Similarly, point mutations are alsocontemplated which may improve therapeutic or productioncharacteristics. These may be achieved by directed or randommutagenesis, as is known in the art, or by thermocycle mutagenicamplification.

In addition, it may be desirable to produce chimeric proteins betweenglutaminase and other proteins. For example, it may be desirable to fusegenes encoding anti-tumor antibodies with the glutaminase gene of thepresent invention. As taught here, covalent complexes of these proteinscan produce dramatic synergistic effects in the arresting of growth oftumor cells. It may provide production benefits to produce suchcomplexes as a chimeric protein, rather than to post-translationallyjoin the two proteins together in vitro. Fusing the glutaminase gene toother genes is also contemplated by the present invention, such asfusing to genes which encode tissue- or tumor-specific ligands, tofacilitate direction of glutaminase to a desired region of the body.

The present invention offers the possibility of obtaining anasparaginase-free glutaminase which may have therapeutic advantages overglutaminase-asparaginase enzymes, since L-asparagine serves as acompetitive inhibitor of glutamine degradation. Elimination ofasparaginase activity from this enzyme may also reduce host toxicity.There are currently no therapeutically useful glutaminases availablewhich lack asparaginase activity.

Inactivating the asparagine binding site of P7A glutaminase withoutaffecting the glutamine binding site can be achieved, since bindingstudies with glutamine and asparagine analogues, DON and DONV,respectively, indicate that the glutamine and asparagine sites are notidentical, though spatially they are close together. DON irreversiblybinds the threonine at amino acid 20, whereas DONV appears to bind to athreonine or serine residue in a different region of the protein. Thecorresponding site-directed mutagenesis of the cloned DNA may be carriedout according to standard techniques (Molecular Cloning, a laboratorymanual-Sambrook et al.-Book 2, 2nd Ed., 1989, pp. 15.80-14.113,Site-directed Mutagenesis of Cloned DNA).

Through oligonucleotide and deletion mutagenesis, an enzyme that isexclusively glutaminase and that is sufficiently small to allow forimproved penetrability of tumors and virus-infected tissue located inthe extravascular space can be obtained. The DNA obtained according toExample 1 may be used. By analysis of the amino acid sequence (asdeduced from the nucleotide sequence) and of X-ray crystallographicdata, regions of the glutaminase protein that are not required forcatalysis or structural integrity can be identified and can be deletedat the DNA level by deleting the relevant nucleotide sequences.

The glutaminase gene of the present invention can be used for transientgene therapy. Generally, the gene can be targeted to and taken up bytransformed cells and expressed in those cells, so that the expressedglutaminase inhibits the growth of the transformed cells. This therapyhas the benefit of avoiding the systemic exposure to the therapeuticagent, which may mitigate potential side-effects. In addition, if thecells are only transiently transfected, as is contemplated, the therapyis reversible.

One method which is contemplated for accomplishing this goal is the useof poly-L-lysine which has been modified with a tissue-specific ligand,as described by Wu et al. J. Biol. Chem. 266:14338-42 (1991). Examplesof such tissue-specific ligands are galactose receptor of the liver,mannose receptor of macrophages, CD4 receptor of helper T cells,epidermal growth factor (EGF) receptor, and thyroid stimulating hormone(TSH) receptor. The glutaminase gene would be placed under thetranscriptional control of a tissue-specific promoter. For example, thepromoters from c-N-ras and c-myc could be used for expression in hepatictumors. These promoters are up-regulated in transformed cells, ascompared to normal cells, and would therefore provide higher levels ofexpression in tumor cells than in normal cells. Plasmids are coated withthe modified poly-L-lysine and injected into the bloodstream of thepatient. The target cells will specifically take up the complexes due tothe tissue-specific ligands. The target cells will specifically expressthe glutaminase construct due to the tissue-specific promoters. Sincecertain neoplasms have been shown to operate at a marginal level ofglutamine availability, and the expression of glutaminase in these cellsfurther depletes the glutamine pool in the tumor cells, the growth ofthese cells is specifically inhibited. Such a treatment should be usefulagainst both fully transformed cells as well as cells in the earlystates of neoplasia, such as early stages of hepatitis B virus (HBV)infection. Since HBV activates expression of c-myc, it would also beexpected to up-regulate this promoter in a glutaminase constructcontrolled by this promoter, leading to high level expression ofglutaminase, which should kill the virus-infected cells.

Another technique for mediating uptake of the PGA gene by transformed orHIV-positive cells utilizes liposomes. (See Nicolau et al., Methods inEnzymology, vol. 149, pp. 157-176 (1987).) Cationic liposomes containinga vector able to express PGA are modified by the addition of specificreceptor ligands or antibodies to the liposome bilayer. (See Hashimotoet al., Methods in Enzymology, vol. 121, pp. 817-829 (1986).) As withthe polylysine method discussed above, liposomes have been used tosuccessfully mediate the specific uptake of foreign DNA in vivo by livercells through the galactose receptor.

Using the combination of cationic liposomes or polylysine as carriers ofa PGA-expressing vector and tissue-specific reagents for targeting,glutaminase can be very specifically and transiently expressed in anytissue or cell of choice. Such tissue-specific reagents for targetingwould include specific antibodies to surface markers and any manner ofligand for any specific cellular receptor. For example, the glutaminasegene can be specifically delivered to CD4+ T cells (the type infected byHIV) in AIDS patients using a portion of the HIV coat protein that bindsthe CD4 antigen, as a ligand on a modified liposome. Carbohydratemodified liposomes containing a PGA expression system can also be used;the galactose receptor of liver, mediates the uptake of such modifiedliposomes. In addition to using ligands for receptors on the cellsurface, antibodies to cell surface markers can be utilized in a similarmanner to deliver these reagents to virtually any cell type.

An additional technique which can be useful in the treatment of patientswith PGA is the use of carrier cells. (See Rosenberg, Cancer Res.(Suppl.). vol. 51, pp. 5074s-5079s (1991).) This technique takesadvantage of the fact that certain T cells have the ability toinfiltrate tumors (tumor infiltrating lymphocytes). These cells aretaken from the cancer patient, transfected with a glutaminase expressionvector, and returned to the patient. Upon return to the body andinfiltration into the neoplasm, a high local level of glutaminase isprovided. Alternatively, a bifunctional carrier can be utilized. Insteadof transfection of the infiltrating lymphocytes with the PGA expressionvector, the vector can be targeted to a specific surface marker on thelymphocytes as detailed above, for example using a monoclonal antibodyin conjunction with poly-L-lysine. The presence of an additionaltargeting ligand (specific for the tumor cells) attached to the vectorallows uptake of the PGA expression vector by the target tumor cells.Essentially the infiltrating lymphocyte is used as a carrier to “drag”the expression vector to the tumor.

Administration of glutaminase to a body can be accomplished by any meansknown in the art. Glutaminase may be directed to particular organs ortissues by administration to arteries which feed the organs or tissuesor by means of an organ or tissue-specific ligand. Direct conjugation ofthe PGA enzyme to functional targeting groups can be employed. Thesegroups include antibodies, lectins, carbohydrates, hormones, peptides,or other compounds that can interact with cell surface molecules. Forspecific examples of this see Methods in Enzymology, vol. 112, pp.238-306 (1985).

Glutaminase can be bound to antibodies, for example, those specific fortumor-associated antigens. A variety of techniques are known forcomplexing two proteins, any of which can be used with glutaminase, solong as enzyme activity and antibody binding capacity are not destroyed.Suitable techniques include those employing the heterobifunctionalreagents SPDP (N-succinimidyl-3-(2-pyridyl dithio)propionate [Carlssonet al., Biochemical Journal, vol. 173, pp. 723-737 (1978)] or SMPT(4-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl dithio)toluene)[Thorpe et al. Cancer Research, vol. 47, pp. 5924-5931 (1987)]. A largenumber of antibodies have been described which are specifically reactivewith tumor-associated antigens. Many are available from the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852,including those to breast, lung and melanoma tumor cells. For a reviewof tumor-specific antibodies see Foon, Cancer Research, vol. 49, pp.1621-1631 (1989).

Tissue culture experiments with Pseudomonas 7A glutaminase (PGA)demonstrate that this enzyme strongly inhibits replication of humanimmunodeficiency virus (HIV) growing in the susceptible human T cellline H9. The presence of 0.4 μg PGA/ml culture medium caused virtually100% inhibition of viral replication and 0.016 μg/ml caused 50%inhibition. A much greater concentration of PGA (50 μg/ml) was requiredto cause noticeable toxicity to the human H9 host cells. Although thetissue culture inhibition has been shown for HIV I-virus, other variantstrains may also be inhibited.

These tissue culture results indicate that PGA is much more toxic to HIVthan to the host human cells and it is expected that PGA will exhibit ahigh therapeutic index when administered to HIV-infected patients.Moreover, in combination with the glutamine antimetabolite DON, PGAproved to be particularly successful in the treatment of lung, breast orcolon cancer. (McGregor, 1989, Proc. Amer. Assoc. Cancer Res. 30:587).

A DNA molecule which codes for a therapeutically suitable glutaminaseand its corresponding polypeptide can be isolated from a microbial,animal or plant cell using oligonucleotide probes prepared according toknown glutaminase protein sequences. Preferably, the DNA is isolatedfrom Pseudomonas 7A cells.

EXAMPLE 1

This example demonstrates the identification of a clone containing thesequence coding for Pseudomonas 7A glutaminase and determination of itsnucleotide sequence.

The glutaminase product is an enzyme that degrades glutamine (an aminoacid that participates in more metabolic processes than any other aminoacid) and therefore hinders growth. In more than two dozen independentexperiments, we had been unable to clone the glutaminase in a variety ofcontexts. We have found it unclonable in high copy number backgroundssuch as pUC. It also proved refractory to cloning in the absence of anupstream transcriptional terminator and a very tightly regulatedpromoter.

Chromosomal DNA was isolated from Pseudomonas 7A (a soil isolateorganism, which has been deposited with the American Type CultureCollection under deposition number ATCC 29598) essentially as described(Strom, 1986, J. Bacteriol. 165:367-372), and was partially digestedwith the restriction enzyme Sau3A. Fragments of this digest averaging5-10 kb were isolated by preparative agarose gel electrophoresis, andcloned into the BamHI site of the vector pBR322. The resultant genomiclibrary (in E. coli strain LE392, ATCC accession no. 33572) was screenedusing mixed oligonucleotide probes (Wallace, et al., 1981, Nucleic AcidsRes. 9:879-89, and Paddock, G.V., 1987, Biotechniques 5:13-16). Partialpeptide sequence information was obtained and used to deduce the threeoligonucleotide probes shown in Table 1. Oligonucleotide probes wereselected for peptide sequence information from the amino terminus of theenzyme (probe A), from the carboxyl terminus (probe C), and from apeptide near the middle of the protein (probe B). Probe B was selectedfor the initial screening of 3560 ampicillin-resistant transformants.From the initial screening two hybridization positive clones wereidentified. These were rescreened using probes A and C. Both cloneshybridized to probe A, but only one of the clones, pME0.5, hybridizedwith probe C. Because pME0.5 had hybridized with all three probes, itwas selected for further analysis.

Crude cell extracts of strain LE392 transformed with plasmid pME0.5 wereprepared by breaking aliquots of an overnight culture, and centrifugingthe homogenate at 15,000 rpm to remove unbroken cells and cell debris.The resulting supernatant was assayed for glutaminase activity by directNesslerization of ammonia (Roberts, J., 1976, J. Biol. Chem.251:2119-2123). To minimize interfering activity by either of the E.coli glutaminase enzymes, the enzyme assay was carried out at pH8.0 andutilizing D-glutamine as the substrate. Neither E. coli enzyme is activeunder these conditions, while the Pseudomonas 7A glutaminase (PGA)retains 87% of its activity (Pruisner, 1976, J. Biol. Chem.,251:3447-3456 and J. Roberts, 1976, J. Biol. Chem. 251:2119-2123).Control experiments with crude cell extracts confirmed the efficacy ofthis assay to measure PGA activity in the absence of E. coli glutaminaseactivity. No activity was found.

TABLE 1 OLIGONUCLEOTIDE PROBES USED FOR DETECTING THE GLUTAMINASE GENEPeptide NH₂-Lys-Glu-Val-Glu-Asn Sequence (1-5) of SEQ ID NO:2 Probe A AA(AG) GA (AG) GT (TCAG) GA (AG) AA (SEQ ID NO:12) (14-mer × 32) PeptideMet-Asn-Asp-Glu-Asn Aln Sequence (161-166 of SEQ ID NO:2 Probe B ATGGA(TC) GA (TC) GA (AG) AT (TCA) (SEQ ID NO:13) GA (AG) (18-mer × 48)Peptide Ile-Phe-Trp-Glu-Tyr-COOH Sequence (332-336 of SEQ ID NO:2) ProbeC AT (TCA) TT (TC) TGGGA (AG) TA (SEQ ID NO:14) (14-mer × 12)

In order to confirm the identity of the putative PGA clone, the regionof homology to the probes used for screening was localized by Southernblot analysis, and the appropriate fragments were partially sequenced.This analysis identified a 1.1 Kb SalI fragment which hybridized toprobe A, and a 1.5 Kb SalI fragment which hybridized to probe B. Thisindicated that there was a SalI site within the gene, and thatsequencing from this site would immediately confirm the identity of thegene as PGA by comparing the nucleotide sequence with the known aminoacid sequence. Sequencing of the 1.1 kb SalI fragment showed that thisfragment encodes the N-terminal 42 amino acids of the glutaminase.

For convenient sequencing, various fragments of the glutaminase codingregion were sub-cloned into a ColE1-based plasmid using standardprotocols Sambrook, et al., supra. These include the 1.1 kb N-terminalSalI fragment (pME1), the 1.5 kb C-terminal SalI fragment (pME2), thethermocyle amplification-mutagenized N-terminus (pME3), and the 200 bpC-terminal PstI fragment (pME11). Numerous sequencing primers weresynthesized using pre-determined glutaminase DNA sequences (see FIG. 1).

Using both the full-length clone, pME0.5, and the sub-cloned genefragments, the glutaminase gene was sequenced in both directions bySanger's chain-termination DNA sequencing method Proc. Natl. Acad. Sci.USA 74:5963 (1977). The purified double-stranded templates weredenatured by the standard alkaline-denaturation method.

The intact coding region (SEQ ID NO:1) encompasses 1008 base pairs andencodes a continuous peptide sequence of 336 amino acids (not includinga 24 amino acid putative signal sequence). The C-terminus is punctuatedby tandem stop codons and a putative transcriptional terminator. Basedon matching this sequence information with the peptide sequencing data,it was concluded that the PGA gene had indeed been cloned.

EXAMPLE 2

This example demonstrates the expression of the gene for Pseudomonas 7Aglutaminase.

Initial experiments showed that even among strong, regulated promoters(e.g. λP_(L))PGA was refractory to overproduction. In order to obtainhigh level controlled expression of the Psuedomonas 7A (P7A) glutaminasein Escherichia coli, we first designed a new vector, pME15 (see FIGS. 3Aand 3B for cloning, and Table 2). The backbone of the vector was pME12(see Table 2) and contains the following features: β-lactamase gene(conferring ampicillin resistance), lac I (repressor of the lactoseoperon), a T7 transcriptional terminator, and a low copy-number ColE1-type origin of replication (pBR322-derived).

TABLE 2 Plasmids Used in Construction of a High-Level Expression PlasmidpME0.5 - genomic clone from a library of Sau3A fragments of P7Achromosomal DNA cloned into the BamHI site of pBR322. This clonecontains the full-length glutaminase gene. pME1 - the N-terminal 1.1 kbSalI fragment of pME0.5 cloned into the SalI site of a Co1E1-basedplasmid. pME2 - the C-terminal 1.5 kb SalI fragment of pME0.5 clonedinto the SalI site of a CO1E1-based plasmid. pME3 - the thermocycleamplification mutagenized front end of the P7A glutaminase containing aBamHI site and an NdeI site at the N-terminus and a BamHI site after thefirst SalI site cloned into a CO1E1-based plasmid. pME4 - pME3 cut withECORV and KpnI, flushed with T4 DNA Polymerase and re-ligated. Thisdeletes the SalI site in the polylinker, leaving the SalI internal tothe glutaminase as unique. pME7 - The 90 bp HinDIII/BamHIPtac-containing fragment resulting from the ligation of overlappingoligonucleotides (Table 3) cloned into pUC19. pME11 - The 200 bp PstIfragment from pME2 which flanks the stop codon of the P7A glutaminasewas cloned into the PstI site of a CO1E1-based plasmid. pME12 - pBR322with the E. coli lacZ gene within the tet gene at the SalI site and theT7 transcriptional terminator at the BamHI site. pME14 - the 1.5 kb SalIfragment from pME2 was cloned into pME4, reconstituting the full-lengthglutaminase gene. pME15 - The HinDIII/EcORI fragment from pME7(containing Ptac) was cloned into pMEl2. pME16 - The BamHI cassette ofpME14 containing the glutaminase gene was cloned into the BamHI site ofpME15. This should give the tac promoter driving glutaminase expression.pME18 - pMEl6 was opened at the unique NdeI site at the N- terminus ofthe glutaminase and filled in with the Klenow fragment of E. coli DNAPolymerase I. This blunt-end product was re-ligated, yielding a distanceof 9 bases between the shine-Dalgarno sequence and the glutaminase startcodon. This should give optimal levels of expression.

TABLE 3 Oligonucleotides Used in Construction of a High Level ExpressionPlasmid Primers for thermocycle amplification mutagenesis of theglutaminase: N-terminus (SEQ ID NO:15) GCCGGATCCA TATGAAGGAA GTGGAGAACCAGCAG Internal SalI site (SEQ ID NO:16) GCGCGGATCC GTCGACGCCA ACCTTGGCAGMutagenized N-terminus of the glutaminase (SEQ ID NOS 17 + 18) GGATCCATATG AAG GAA GTG GAG AAC         Met Lys Glu Val Glu Asn . . .Oligonucleotide for tac promoter top (SEQ ID NO:19): AGCTTACTCCCCATCCCCCT GTTGACAATT AATCATCGGC TC GTATAATGTG TGGAATTGTG AGCGGATAACAATTTCACAC AGGAAACAG bottom (SEQ ID NO:20) GATCCTGTTT CCTGTGTGAAATTGTTATCC GCTCACAATT CCACACA TTATACGAGC CGATGATTAA TTGTCAACAGGGGGATGGGG AGTA Filled in product of pME18 (SEQ ID NOS 21 + 22)           lacO S.D. S.D. 5′ AATTGTGAGCGGATAACAATTTCACAC AGGA AACAGGATCCATAT ATG AAG             Met Lys GAA GTG GAG AAC 3′ Glu Val GluAsn . . .

We chose as our promoter the tac (hybrid trp/lac) promoter containing alac operator sequence (conferring susceptibility to repression by lacI). (deBoer, et al., Proc. Natl. Acad. Sci. USA, 80:21 (1983), andRussel, et al., Gene 20:231 (1982).) We synthesized overlappingoligonucleotides which were ligated and cloned as a BamHI/HindIIIfragment into the cloning vector pUC19, resulting in pME7. TheBamHI/EcoRI fragment of pME7 was cloned into pME12 to form pME15. Thisprovides the tac promoter controlled by lac I and hence, it is induciblewith isopropyl-β-thio-D-galactoside (IPTG). This promoter is active inthe same orientation as the unidirectional origin of replication andwill therefore not interfere with plasmid propagation. A transcriptionalterminator is present immediately upstream of the transcriptional startsite, eliminating “read-through” transcription. The combination ofcontrol by lac I and an upstream transcriptional terminator provides avector with the ability to stably propagate and express even the toxicglutaminase genes. Additionally, having a plasmid-encoded lac I genealso allows for virtual host independence.

As we have previously observed, the full length P7A glutaminase ispresent on two SalI fragments: a 1.1 kb fragment containing theN-terminus of the gene and a 1.5 kb fragment containing the C-terminus.In order to clone this gene into an expression system, we wished toengineer the sequence at the N-terminus to provide convenient sites forrestriction endonuclease. This would allow the cloning of the gene intonumerous established expression vectors in addition to our vector,pME15. For this reason, we used mutagenic thermocycle amplificationprimers (see Table 3) to generate a BamHI site and an NdeI site at theN-terminal lysine residue. This mutagenesis also adds a methionineresidue immediately upstream of the N-terminal lysine. We also added aBamHI site after the internal SalI site.

The thermocycle amplification-mutagenized N-terminal fragment of theglutaminase gene was cloned into a ColE1-based vector as a BamHIfragment (pME3). The polylinker between KpnI and EcoRV was deletedremoving the endogenous SalI site, generating pME4. The full-lengthglutaminase was reconstituted by ligation of the 1.5 kb SalI fragment,encoding the C-terminus, into the unique SalI site of pME4, yieldingpME14. The 1.7 kb BamHI fragment from pME14 was cloned into theexpression vector pME15. This clone (pME16) provides glutaminaseexpression driven by the tac promoter. In an attempt to achieve higherlevels of expression, we opened pME16 at the NdeI site and filled it inusing the Klenow fragment of E. coli DNA Polymerase I. Upon re-ligation,the spacing between the Shine-Dalgarno sequence and the translationstart-site became optimal (see Table 3).

This vector (pME18) is stable and directs expression of authentic P7Aglutaminase. It has been deposited at the American Type CultureCollection under the terms of the Budapest Treaty on Nov. 3, 1992, andassigned the accession no. 69117. For the purpose of protein production,cells were grown to mid-log phase at 37° C. and treated with 0.4 mM IPTGfor 1 -6 hours. Protein production was monitored by denaturingpolyacrylamide gel electrophoresis (FIG. 4) and glutaminase specificactivity. Such culturing has yielded activities as high as 4100 U/literof culture, representing approximately 3% of the total cellular protein.Since Pseudomonas P7A produces only 350 U/liter, this represents a12-fold increase in enzyme production.

In order to assure that the observed activity was contributed by thePseudomonas glutaminase as opposed to endogenous E. coli glutaminase Aand B, the reaction was repeated with D-glutamine at pH 8. OnlyPseudomonas enzyme functions under these conditions, in fact, itconverts D-glutamine to glutamate 87% as efficiently as it convertsL-glutamine. The results demonstrate that all the measurable activitywas contributed by PGA. Bradford protein assays were also done on thecrude cell extracts to allow calculation of the enzyme activity asspecific activity. Samples of each extract were also analyzed onSDS-PAGE gels essentially as described by Laemmli (Laemmli, U.K. (1970)Nature (London) 227:680-685). The resulting enzyme induction curve isshown in FIG. 5, where the increase in glutaminase activity in the cellextract using D-glutamine as substrate is shown. As can be seen, theactivity of the enzyme utilizing D-glutamine as a substrate increasesover 4000-fold after IPTG induction, while control culture showsvirtually no increase in D-glutaminase activity.

For the sake of efficient translation of the glutaminase in E. coli, anN-terminal methionine codon was added (see Table 3). It was of someconcern whether or not this extra amino acid would alter the activity ofthe enzyme. To test this, we measured enzyme activity against L- andD-glutamine as well as L- and D-asparagine. The ratios of activitybetween the L- and D-isomers were the same for both the native and theengineered enzyme (e.g., L-:D-glutamine and L-:D-asparagine). Anotherconcern was that this alteration might adversely effect the in vivohalf-life. To test this, we performed in vivo half-life studies in mice;both enzymes showed the same in vivo half-life. Based on these combineddata, we conclude that the extra N-terminal methionine residue does notalter the biological activity of the enzyme.

EXAMPLE 3

This example demonstrates the use of P7A glutaminase sequences toidentify homologous sequences in other bacterial species.

Chromosomal DNA from Pseudomonas aeruginosa and Achromobacter sp. wasisolated using standard protocols. After complete digestion with EcoRI,DNA fragments were resolved on a 30 cm, 1% agarose gel at 50V for 15hours in 89 mM Tris-Cl, pH8; 89 mM Borate; and 1 mM EDTA. Transfer andhybridization were as described (Maniatis et al. Molecular cloning: Alaboratory Manual, pp. 382-389, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. 1982) using stringent conditions. The probe was thecoding region of the P7A glutaminase gene labeled with α-³²Pdeoxycytosine triphosphate. Lane 1, Pseudomonas 7A (2 hr. exposure);lane 2, Pseudomonas aeruginosa (6 hr. exposure); lane 3, Achromobactersp. (24 hr. exposure). Results are shown in FIG. 6.

EXAMPLE 4

This example demonstrates in vitro inhibition of human melanoma cells byglutaminase linked to anti-melanoma antibody.

Human melanoma cells (Heatherington) were incubated in vitro for 30minutes with either free R24 anti-melanoma antibody, free glutaminase,or covalently bound antibody-glutaminase complex. The covalently boundantibody-glutaminase complex was prepared utilizing theheterobifunctional reagent SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate [Carlsson et al., Biochemical Journal, vol. 173, pp.723-737 (1978)]. The cells were washed and placed in fresh tissueculture media, and ³H-Thymidine incorporation was measured. Theincorporation of the melanoma cells is shown in Table 4. Neither freeantibody or free glutaminase inhibited thymidine incorporation. Onlycells incubated with the antibody-glutaminase complex exhibitedinhibition of ³H-Thymidine incorporation. Thus, the two components,antibody and glutaminase act synergistically to inhibit tumor cellgrowth.

TABLE 4 % INHIBITION OF ³H- THYMIDINE TREATMENT INCORPORATION FreeAntibody 0 Free Glutaminase (0.06 IU/ml) 0 Antibody-Glutaminase (0.06IU/ml) Complex 93 Relevant cultures were deposited at the American TypeCulture Collection, located at 10801 University Boulevard. Manassas, VA20110-2209. Pseudomonas sp., 7A was deposited on March 3, 1992 -Accession Number 29598. Escherichia Coli, pME18, was deposited onNovember 3, 1992 - Accession Number 69117.

22 1017 base pairs nucleic acid double linear DNA (genomic) not providedCDS 1..1011 1 AAG GAA GTG GAG AAC CAG CAG AAG CTG GCC AAC GTG GTG ATCCTC GCC 48 Lys Glu Val Glu Asn Gln Gln Lys Leu Ala Asn Val Val Ile LeuAla 1 5 10 15 ACC GGC GGC ACC ATC GCC GGC GCT GGC GCC AGC GCG GCC AACAGC GCC 96 Thr Gly Gly Thr Ile Ala Gly Ala Gly Ala Ser Ala Ala Asn SerAla 20 25 30 ACC TAC CAG GCT GCC AAG GTT GGC GTC GAC AAG CTG ATT GCC GGCGTG 144 Thr Tyr Gln Ala Ala Lys Val Gly Val Asp Lys Leu Ile Ala Gly Val35 40 45 CCG GAG CTG GCC GAC CTG GCC AAT GTG CGC GGC GAG CAG GTG ATG CAG192 Pro Glu Leu Ala Asp Leu Ala Asn Val Arg Gly Glu Gln Val Met Gln 5055 60 ATC GCC TCC GAA AGC ATC ACC AAC GAC GAC CTG CTC AAG CTG GGC AAG240 Ile Ala Ser Glu Ser Ile Thr Asn Asp Asp Leu Leu Lys Leu Gly Lys 6570 75 80 CGC GTG GCC GAG CTG GCC GAC AGC AAT GAC GTC GAT GGC ATC GTC ATC288 Arg Val Ala Glu Leu Ala Asp Ser Asn Asp Val Asp Gly Ile Val Ile 8590 95 ACC CAT GGC ACC GAC ACC CTG GAA GAA ACC GCC TAC TTT TTG AAC CTC336 Thr His Gly Thr Asp Thr Leu Glu Glu Thr Ala Tyr Phe Leu Asn Leu 100105 110 GTG GAA AAG ACC GAC AAG CCG ATC GTC GTG GTC GGT TCC ATG CGC CCC384 Val Glu Lys Thr Asp Lys Pro Ile Val Val Val Gly Ser Met Arg Pro 115120 125 GGC ACC GCC ATG TCC GCC GAC GGC ATG CTC AAC CTG TAC AAC GCC GTG432 Gly Thr Ala Met Ser Ala Asp Gly Met Leu Asn Leu Tyr Asn Ala Val 130135 140 GCC GTG GCC AGC AAC AAG GAC TCG CGC GGC AAG GGC GTG CTG GTG ACC480 Ala Val Ala Ser Asn Lys Asp Ser Arg Gly Lys Gly Val Leu Val Thr 145150 155 160 ATG AAC GAC GAG ATC CAG TCC GGG CGT GAC GTG AGC AAG TCG ATCAAC 528 Met Asn Asp Glu Ile Gln Ser Gly Arg Asp Val Ser Lys Ser Ile Asn165 170 175 ATC AAG ACC GAA GCC TTC AAG AGC GCC TGG GGC CCG CTG GGC ATGGTG 576 Ile Lys Thr Glu Ala Phe Lys Ser Ala Trp Gly Pro Leu Gly Met Val180 185 190 GTG GAA GGC AAG TCG TAC TGG TTC CGC CTG CCG GCC AAG CGC CACACG 624 Val Glu Gly Lys Ser Tyr Trp Phe Arg Leu Pro Ala Lys Arg His Thr195 200 205 GTC AAC TCC GAG TTC GAC ATC AAG CAG ATC AGC AGC CTG CCC CAGGTG 672 Val Asn Ser Glu Phe Asp Ile Lys Gln Ile Ser Ser Leu Pro Gln Val210 215 220 GAC ATC GCC TAC AGC TAT GGC AAC GTC ACC GAC ACG GCC TAC AAGGCC 720 Asp Ile Ala Tyr Ser Tyr Gly Asn Val Thr Asp Thr Ala Tyr Lys Ala225 230 235 240 CTG GCA CAG AAC GGC GCC AAG GCG CTG ATC CAT GCC GGC ACCGGC AAT 768 Leu Ala Gln Asn Gly Ala Lys Ala Leu Ile His Ala Gly Thr GlyAsn 245 250 255 GGC TCG GTG TCG TCG CGG GTG GTG CCA GCC CTG CAG GAG CTGCGC AAG 816 Gly Ser Val Ser Ser Arg Val Val Pro Ala Leu Gln Glu Leu ArgLys 260 265 270 AAC GGC GTG CAG ATC ATT CGT TCG TCC CAC GTC AAC CAG GGCGGT TTC 864 Asn Gly Val Gln Ile Ile Arg Ser Ser His Val Asn Gln Gly GlyPhe 275 280 285 GTG CTG CGT AAC GCC GAG CAG CCC GAC GAC AAG AAC GAC TGGGTC GTG 912 Val Leu Arg Asn Ala Glu Gln Pro Asp Asp Lys Asn Asp Trp ValVal 290 295 300 GCC CAC GAC CTG AAC CCG CAG AAG GCC CGC ATC CTG GCG ATGGTG GCA 960 Ala His Asp Leu Asn Pro Gln Lys Ala Arg Ile Leu Ala Met ValAla 305 310 315 320 ATG ACC AAG ACC CAG GAC AGC AAG GAG CTG CAG CGC ATTTTC TGG GAA 1008 Met Thr Lys Thr Gln Asp Ser Lys Glu Leu Gln Arg Ile PheTrp Glu 325 330 335 TAC TGATAA 1017 Tyr 337 amino acids amino acidlinear protein not provided 2 Lys Glu Val Glu Asn Gln Gln Lys Leu AlaAsn Val Val Ile Leu Ala 1 5 10 15 Thr Gly Gly Thr Ile Ala Gly Ala GlyAla Ser Ala Ala Asn Ser Ala 20 25 30 Thr Tyr Gln Ala Ala Lys Val Gly ValAsp Lys Leu Ile Ala Gly Val 35 40 45 Pro Glu Leu Ala Asp Leu Ala Asn ValArg Gly Glu Gln Val Met Gln 50 55 60 Ile Ala Ser Glu Ser Ile Thr Asn AspAsp Leu Leu Lys Leu Gly Lys 65 70 75 80 Arg Val Ala Glu Leu Ala Asp SerAsn Asp Val Asp Gly Ile Val Ile 85 90 95 Thr His Gly Thr Asp Thr Leu GluGlu Thr Ala Tyr Phe Leu Asn Leu 100 105 110 Val Glu Lys Thr Asp Lys ProIle Val Val Val Gly Ser Met Arg Pro 115 120 125 Gly Thr Ala Met Ser AlaAsp Gly Met Leu Asn Leu Tyr Asn Ala Val 130 135 140 Ala Val Ala Ser AsnLys Asp Ser Arg Gly Lys Gly Val Leu Val Thr 145 150 155 160 Met Asn AspGlu Ile Gln Ser Gly Arg Asp Val Ser Lys Ser Ile Asn 165 170 175 Ile LysThr Glu Ala Phe Lys Ser Ala Trp Gly Pro Leu Gly Met Val 180 185 190 ValGlu Gly Lys Ser Tyr Trp Phe Arg Leu Pro Ala Lys Arg His Thr 195 200 205Val Asn Ser Glu Phe Asp Ile Lys Gln Ile Ser Ser Leu Pro Gln Val 210 215220 Asp Ile Ala Tyr Ser Tyr Gly Asn Val Thr Asp Thr Ala Tyr Lys Ala 225230 235 240 Leu Ala Gln Asn Gly Ala Lys Ala Leu Ile His Ala Gly Thr GlyAsn 245 250 255 Gly Ser Val Ser Ser Arg Val Val Pro Ala Leu Gln Glu LeuArg Lys 260 265 270 Asn Gly Val Gln Ile Ile Arg Ser Ser His Val Asn GlnGly Gly Phe 275 280 285 Val Leu Arg Asn Ala Glu Gln Pro Asp Asp Lys AsnAsp Trp Val Val 290 295 300 Ala His Asp Leu Asn Pro Gln Lys Ala Arg IleLeu Ala Met Val Ala 305 310 315 320 Met Thr Lys Thr Gln Asp Ser Lys GluLeu Gln Arg Ile Phe Trp Glu 325 330 335 Tyr 20 base pairs nucleic acidsingle linear other nucleic acid /desc = “Primer” not provided 3TGCAGCTTGA GCAGGTCGTC 20 21 base pairs nucleic acid single linear othernucleic acid /desc = “Primer” not provided 4 CTGGCCGACC TGGCCAATGT G 2120 base pairs nucleic acid single linear other nucleic acid /desc =“Primer” not provided 5 CCTACTTTTT GAACCTCGTG 20 20 base pairs nucleicacid single linear other nucleic acid /desc = “Primer” not provided 6CAAGTCGTAC TGGTTCCGCC 20 21 base pairs nucleic acid single linear othernucleic acid /desc = “Primer” not provided 7 CAATCGTCCT GGCGACTCGT G 2120 base pairs nucleic acid single linear other nucleic acid /desc =“Primer” not provided 8 GCAGATCATT CGTTCGTCCA 20 20 base pairs nucleicacid single linear other nucleic acid /desc = “Primer” not provided 9TGACGATGCC ATCGACGTCA 20 20 base pairs nucleic acid single linear othernucleic acid /desc = “Primer” not provided 10 TCACGTCACG CCCGGACTGG 2020 base pairs nucleic acid single linear other nucleic acid /desc =“Primer” not provided 11 AGCTCCTGCA GGGCTGGCAC 20 14 base pairs nucleicacid single linear other nucleic acid /desc = “Oligonucleotide probe”not provided 12 AARGARGTNG ARAA 14 18 base pairs nucleic acid singlelinear other nucleic acid /desc = “Oligonucleotide probe” not provided13 ATGGAYGAYG ARATHGAR 18 14 base pairs nucleic acid single linear othernucleic acid /desc = “Oligonucleotide probe” not provided 14 ATHTTYTGGGARTA 14 35 base pairs nucleic acid single linear other nucleic acid/desc = “Oligonucleotide” not provided 15 GCCGGATCCA TATGAAGGAAGTGGAGAACC AGCAG 35 30 base pairs nucleic acid single linear othernucleic acid /desc = “Oligonucleotide” not provided 16 GCGCGGATCCGTCGACGCCA ACCTTGGCAG 30 26 base pairs nucleic acid single linear othernucleic acid /desc = “Oligonucleotide” not provided CDS 9..26 17GGATCCAT ATG AAG GAA GTG GAG AAC 26 Met Lys Glu Val Glu Asn 1 5 6 aminoacids amino acid linear protein not provided 18 Met Lys Glu Val Glu Asn1 5 91 base pairs nucleic acid single linear other nucleic acid /desc =“Oligonucleotide” not provided 19 AGCTTACTCC CCATCCCCCT GTTGACAATTAATCATCGGC TCGTATAATG TGTGGAATTG 60 TGAGCGGATA ACAATTTCAC ACAGGAAACA G91 91 base pairs nucleic acid single linear other nucleic acid /desc =“Oligonucleotide” not provided 20 GATCCTGTTT CCTGTGTGAA ATTGTTATCCGCTCACAATT CCACACATTA TACGAGCCGA 60 TGATTAATTG TCAACAGGGG GATGGGGAGT A91 63 base pairs nucleic acid single linear other nucleic acid /desc =“Oligonucleotide” not provided CDS 46..63 21 AATTGTGAGC GGATAACAATTTCACACAGG AAACAGGATC CATAT ATG AAG GAA 54 Met Lys Glu 1 GTG GAG AAC 63Val Glu Asn 5 6 amino acids amino acid linear protein not provided 22Met Lys Glu Val Glu Asn 1 5

We claim:
 1. An E. coli cell which comprises a gene that encodes atherapeutically suitable glutaminase.
 2. The cell of claim 1 which isdeposited at the ATCC as accession no.
 69117. 3. The cell of claim 1which is capable of expressing said gene.
 4. The cell of claim 1 whereinsaid gene has the nucleotide sequence shown in SEQ ID NO:1.
 5. Anisolated and purified DNA molecule comprising a nucleotide sequencecoding for a therapeutically suitable glutaminase.
 6. The DNA moleculeof claim 5 wherein said glutaminase has a K_(m) of 10⁻⁶ to 10⁻⁴ M forits reactants and remains active in human sera.
 7. The DNA molecule ofclaim 5 wherein said glutaminase is a Pseudomonas glutaminase.
 8. TheDNA molecule of claim 5 wherein said glutaminase is a Pseudomonas 7Aglutaminase.
 9. The DNA molecule of claim 5 which comprises thenucleotide sequence shown in SEQ ID NO:1.
 10. The DNA molecule of claim6 wherein said molecule is capable of expressing said glutaminase in arecombinant cell.
 11. The DNA molecule of claim 5 wherein saidglutaminase has the sequence shown in SEQ ID NO:2.
 12. The DNA moleculeof claim 6 wherein expression of said glutaminase is controlled by aninducible promoter.
 13. The DNA molecule of claim 6 wherein expressionof said glutaminase is controlled by a repressor.
 14. The DNA moleculeof claim 6 wherein the promoter is tac.
 15. The DNA molecule of claim 10comprising a transcriptional terminator 3′ and 5′ to said sequence ofglutaminase.
 16. The DNA molecule of claim 8 having a methionine codon5′ to the initial lysine codon of mature glutaminase.
 17. A cellaccording to claim 1, which comprises a Pseudomonas 7Aglutaminase-asparaginase gene.